Small-scale cell culture devices have existed in the form of shaker flasks and roller bottles for years. More recently, single layer and now multilayered flask bottles have been developed. The current trend in research has demanded more cells to fulfill the high throughput screen campaigns in drug discovery companies. Additionally, the use of cell-based assays are rapidly increasing because of the push to challenge potential drug candidates earlier in the development process. A typical cell based assay or HTS screening run can require from about 109 to about 1010 cells. A standard single layered flask can deliver about 107 cells. The current cell need requires researchers to maintain and feed 10 to 100 standard flasks in order to reach a requisite number of cells to run a given cell based assay or HTS screening, so new multilayered formats are needed. Thus, it would be desirable to have a cell culture system that provides a high number of cells without a substantial increase in the number of standard flasks to be maintained and fed.
One such product has two trays attached to each other and inserted within a flask to form a three culture layered flask. The flask is generally rectangular with a typical threaded bottle opening at its forward most converging sidewall which is in the form of bottle neck (See U.S. Pat. No. 5,310,676). Each tray has a sidewall around its entire periphery and the sidewalls are sealed to each other. In order to provide cell and liquid access to the middle and lower tray one needs to build a tunnel along the end wall of the trays or flask that liquidly interconnect the trays so that liquid and cells can be flowed into and out of the trays.
Another product uses up to ten trays having each upper surface covered by a gas permeable film. Each tray has a sidewall extending upwardly from the surface. The trays are stacked together and the sidewalls fused to form an integral mass. A manifold and bottle neck sidewall with a typical threaded bottle opening is then bonded to the front end of the trays to complete the flask. (See WO 2008/069902 A3). The manifold provides access to the opened end of the trays.
The systems taught in U.S. Pat. No. 5,310,676 and WO 2008/069902 A3) each have drawbacks. Neither is easily accessible with a pipette or syringe for the addition of fluid or cells, sampling or removal of the cells upon completion of the growth cycle. In fact, the use of a pipette or syringe is restricted because the bottle opening is positioned on the sidewall and the trays are blocking pipette access to the media and the cells. It would be desirable to be able to pipette the media and cells directly from a culture device, using standard pipetting tools and standard cell culture techniques.
Application of cells and liquid is difficult and often incomplete. U.S. Pat. No. 5,310,676 relies upon the molded in tunnel to aid in its distribution which can be difficult and time consuming. Liquid must be added to the flask in an upright position which can lead to foaming and damage to critical proteins in the media. Then the flask is rotated in various directions to ensure that all trays receive an adequate amount of fluid. Likewise, removal of the liquid and/or cells require passage through the tunnel in order to be recovered. Lastly, the number of trays is insufficient to effectively increase the high yields needed by today's scientists.
WO 2008/069902 uses a manifold to distribute liquid and cells into a flask by adding the liquid (containing cells and/or growth media) to the neck portion of the flask. The neck is closed and the flask is shaken tapped, or otherwise moved to dislodge any air that would become trapped within the layers of the system. Lastly as the space between the trays is very small and the gas permeable film forms the upper limit of media on any given tray, the thickness of the media is fixed at about 0.32 mls per cm2. Depending on the cell types being cultured the media needs are different. For example, a human or mouse stem cell is a highly metabolically active cell and demands a high level of media, typically in the range of 0.4 mls per cm2, whereas a slow metabolizing cell line such as CHO, MDCK or fibroblast may only require 0.2 mls per cm2. Since WO 2008/069902 has a fixed media volume to fill the system, the researcher needs to adjust the cell feeding, and maintain a schedule for each cell type being cultured, or else waste expensive media and additives. Additionally, scientists prefer to be able to vary the amount of media they can use in order to optimize the growing conditions, a parameter that WO 2008/069902 does not enable a scientist to optimize. It would be desirable to provide a culture device having layers that easily fill with media without the restriction of tunnels, or the need to dislodge air entrapped within the layers.
As the cells grow, the researcher routinely needs to access the status of growth of the culture so that appropriate steps can be taken, such as when the cells are near confluent the researcher will detach them from the culture tray for use in assays, screens or the like, or to reseed new culture systems to continue to expand the cell line. It is important to recover the cells prior to 100% confluency. These systems typically require the researcher to move the culture system to an expensive microscope that may or may not be in the culture area, to investigate the cell growth status. It would desirable to be able to fill the culture device with the desired amount of media and additives to satisfy the needs of the cell type, as well as the researcher's work schedule. Additionally, it would be desirable to provide a cell culture system that enables visualization of the cells without having to transport the flask or the like housing the cells to an expensive microscope.
Typically, laboratories that are working with high numbers of cells as described, use a variety of automated equipment such as multiwell plate handlers and liquid dispensing systems to increase throughput and reduce data variability due to the operator error. These culture systems can be automated, but are limited to systems equipped with articulating arms that can grasp the system and pour the liquid out into a receiver vessel. Systems with these complex articulating arms are expensive and not available to most laboratories. Therefore, most uses for the above culture systems are limited to a manual operator manipulating the systems, which runs counter to the work practices of high throughput laboratories. Thus, it would be desirable to provide a cell culture system compatible with the automated systems currently in most laboratories, and not require special articulating arms to automate.